DESCRIPTION: Biochemical and physiological studies of the mechanisms underlyi taste transduction suggest that bitter and sweet stimuli are transduced by G-protein-coupled receptors. Five different G proteins are expressed in taste buds (which contain both receptor and non-receptor cell types), and one G-protein, gustducin, is expressed only in taste receptor cells. The responses bitter and sweet stimuli were suppressed in gustducin-knockout mice, although not completely, suggesting that gustducin has some role in these responses. T sequence of gustducin is similar to that of transducin, the photoreceptor G-protein, and both G-proteins can activate a phosphodiesterase from taste tissu however, existing data suggest that this action of gustducin is unlikely to be the primary transduction pathway for either bitter or sweet stimuli. The role of the gustducin-mediated pathways in taste cells remain uncertain. In additi and despite considerable effort, no taste receptors that couple through gustducin or any other G-protein have been cloned; however, bovine rhodopsin which normally couples through transducin, will activate gustducin. The goal of this research application is to generate transgenic mice that express bovine rod opsin in their gustducin-expressing taste receptor cells. Using these engineered taste cells, the taste transduction pathways that invol gustducin will be studied. To do this, methods would be employed that are similar to those used earlier by the applicant to express alpha-galactosidase and Green Fluorescent Protein in the gustducin lineage of taste cells. Taste buds from the transgenic animals would be isolated and incubated in the dark with 11-cis-retinal to regenerate rhodopsin. Then, bright light would be used as a stimulus to activate the receptor. With sufficient light intensity, it i expected that a gustducin-mediated response could be elicited which is wholly dependent upon the endogenous elements in the taste transduction pathway downstream of the receptor. Electrophysiological studies would then be conducted to identify the membrane conductance changes elicited as a consequen of activating this pathway. The strategy of generating the transgenic animals should not discriminate between sweet-responsive and bitter-responsive taste cell phenotypes containin gustducin, so that all the sweet- and bitter-responsive cell types should be represented in the opsin-expressing population. To distinguish between the effects mediated by the gustducin transduction pathways in the two phenotypes, it will be necessary first to identify the gustducin/opsin-containing cells. This will be done by co-expressing Green Fluorescent Protein with bovine opsin to provide a visible marker. Second, the normal phenotypes of cells can be assessed by testing with sweeteners or the very bitter denatonium. Finally, t gustducin-mediated portion of the normal response could be tested by stimulati rhodopsin with light.